Interleukin 7 inhibit autophagy via P53 regulated AMPK/mTOR signaling pathway in non-small cell lung cancer

Interleukin 7 (IL-7) has been demonstrated regulating lymphangiogenesis, apoptosis, and proliferation. Whether IL-7 induce or inhibit autophagy in non-small cell lung cancer (NSCLC) are unknown. In this study, Western blot was used to detect cytoplasmic and nuclear protein of p53, total protein of AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR) and Light chain 3 (LC3). Quantitative Real-Time PCR (qRT-PCR) was used to detect p53 mRNA level after treated with IL-7. Then using transmission electron microscopy to observe the morphological change of autophagosome. 123 cases of NSCLC were collected for survival analysis, immunohistochemistry staining and cox regression multivariate analysis. We find that IL-7 induce the p53 translocation from nucleus to cytoplasm, then IL-7 down-regulate phosphorylation of AMPK and up-regulate phosphorylation of mTOR. The expression of AMPK and p53 were associated with IL-7/IL-7R and mTOR expression. Clinically, AMPK and p53 were well correlated with stage and survival of lung cancer patients. IL-7R, mTOR and tumor stage were the strongest predictors of survival. In conclusion, IL-7 inhibit autophagy in NSCLC via P53 regulated AMPK/mTOR signaling pathway. AMPK and p53 are correlated with patients’ survival. IL-7R, mTOR and tumor stage are the strongest predictor of survival.


Western blot analysis.
After IL-7 stimulation, total protein were extracted with NP-40 lysis buffer (Beyotime Biotechnology, Shanghai, China) (containing 1× protease and phosphatase inhibitors cocktail) on ice for 25 min. Then centrifuged at 12,000 rpm for 20 min at 4 •C. After quantified, total protein sample (40 μg protein per lane) were electrophoresed on 8% and 12% SDS polyacrylamide electrophoresis (SDS-PAGE) gel. Before transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA), gel were cropped for each specific antibody according to their protein molecular weight. Then, membrane were incubated with specific primary antibody in 4 °C overnight and secondary antibody for 2 h at room temperature. Finally, membrane were visualized using electrochemiluminescence (ECL) (Wanlei, Shenyang, China) and detected with BioImaging System (UVP Inc., Upland, CA, USA).
Cytoplasmic and nuclear protein were extracted by using cytoplasmic and nuclear protein extraction kit (Beyotime Biotechnology, Shanghai, China) according to manufacturer' s protocol. Nuclear protein (30 μg) and cytoplasmic protein (30 μg) were electrophoresed on 8% SDS-PAGE gel then transferred to PVDF membrane. Other process are similar as previously described.
RT-qPCR. Sample were isolated by trizol, then 1 μg RNA were reverse transcribed into single-strand cDNA using PrimeScript RT reagent kit (Takara, Tokyo, Japan, RR047A). Finally SYBR Green Real Time PCR master mix (Takara, Tokyo, Japan, RR820A) was used to perform in 7900HT Fast Real-Time PCR System (Applied Bio-Patients and specimens. A total of 123 cases of NSCLC were obtained from the 1st January 2010 to the 31st December 2020 at General Hospital of Northern Theater Command, Shenyang, China. Study was approved by General Hospital of Northern Theater Command Ethics Committee. The tumour tissues in this study were from patients who had NSCLC proved by pathological diagnosis without distant metastasis. None of the 123 cases had received radiation therapy or chemotherapy before surgery. The TNM staging system of the UICC (2017) was used to classify the specimens.
The survival time was calculated from the operation day to death via the evaluation of recurrence and metastasis or until the last follow-up date (December 2013). The following-up of the surviving patients averaged 27.09 months and ranged from 1 to 156 months. The study has been approved by the General Hospital of Northern Theater Command Ethics Committee.
Immunohistochemistry. Four-micron thick sections were prepared from the paraffin-embedded tissues.
All the samples were evaluated by two independent pathologists. The staining intensity and extent of IL-7, IL-7R, P53, AMPK, and mTOR were scored as follows. Staining intensity was defined as the depth of shade of the protein and graded as 0 (no staining), 1 (mild staining), 2 (moderate staining), 3 (strong staining). Staining extent was defined as the percentage of tumor cells with positive brown-yellow staining and graded as follows: 0 (< 10%), 1 (10-49%), 2 (≥ 50%). Then sum index of the two variables was obtained and a total score of 4 was considered as the point to categorize the staining as high (≥ 4) or low (< 4) 11-13 . Statistical analysis. SPSS19.0 (SPSS incorporated, Chicago) was used for all analysis. One-way analysis of variance (ANOVA) was performed for statistical analysis, and post hoc analysis Dunnett's test was used for each group compared to control. P-value < 0.05 was considered statistically significant. All the experiments were conducted in triplicate. The Chi-square test was used to analyze the relationship between P53 and AMPK expression and clinical pathological factors; these tests were also used to evaluate whether there was any significant relationship between P53/AMPK and IL-7/IL-7R; Kaplan-Meier curves were used for survival analysis, and logrank was determined based on the differences; Cox regression multivariate analysis was used to evaluate P53 and AMPK as prognostic factors to compare them with other strong prognostic factors for prognosis in lung cancer.
Ethics approval. The study has been approved by the General Hospital of Northern Theater Command Ethics Committee.
Approval for human experiments. 1 Results IL-7 induced the p53 translocation from nucleus to cytoplasm. We isolated nuclear and cytoplasmic protein of A549 at 8 h, 12 h and 24 h (Fig. 1a). IL-7 exert its peak effect at 12 h, thus we chose 12 h as optimal incubation time in our sequential experiments. After IL-7 treated for 12 h, we observed a notably increase of cytoplasmic p53 (Fig. 1a), so as an escalation of p53 mRNA level (Fig. 1c). This effect could be blocked by anti-IL-7 receptor antibody (A-IL-7R) (Fig. 1b). Cytosolic Lamin B and nucleus GAPDH protein expression almost negative illustrate cytoplasmic and nuclear protein extraction kit is viable and our application method is correct (Fig. 1a,b). These data suggest IL-7 affected p53 distribution between nuclear and cytoplasm in A549 cell line.

IL-7 affect phosphorylated form of AMPK and mTOR.
We detected total protein of A549 cells after incubated with IL-7 and IL-7 plus its A-IL-7R antibody: the result show that p-AMPK decreased and p-mTOR increased, while total protein of AMPK and mTOR did not change, and this effect could be blocked by A-IL-7R, which indicate that IL-7 affect p-AMPK and p-mTOR (Fig. 2). www.nature.com/scientificreports/ IL-7 regulate mTOR via AMPK. We use Compound C (AMPK inhibitor) and Rapamycin (mTOR inhibitor) to investigate the relationship between AMPK and mTOR. After treated with Rapamycin, in Western Blot analysis, seldom do we observe the change of p-AMPK protein (Fig. 3a), but we observed a dramatically p-mTOR suppression (Fig. 3a), which indicate mTOR is not the up-regulator for AMPK. Then, cells were treated with Compound C, results show that Compound C shut down the p-AMPK/AMPK expression and escalate the p-mTOR/mTOR expression level (Fig. 3b). All these results indicate that AMPK is the upstream factor for mTOR and IL-7 regulate mTOR via AMPK. www.nature.com/scientificreports/ IL-7 regulate AMPK and mTOR via p53. Firstly, We added PFT-α (p53 inhibitor) to further explore relationship between p53, AMPK and mTOR. As shown in (Fig. 4a,e), IL-7 manipulate an increase of cytoplasmic p53 protein and p53 mRNA level, however there is no obvious change in p53 nuclear protein and these effect can be blunt by PFT-α (Fig. 4a). Meanwhile, PFT-α cause a significant up-regulation of p-AMPK and downregulation of p-mTOR (Fig. 4b). Then, rapamycin was used to observe the change of cytoplasmic and nuclear p53 protein. As shown in Fig. 4c, seldom do we observe change neither nuclear p53 protein nor cytoplasmic p53 protein, which indicate that mTOR is not the upstream regulator for p53. Last bu not least, Compound C was used, western blot results show that Compound C can not regulate p53 protein neither nucleus nor cytoplasm (Fig. 4d), which demonstrate AMPK is not the up-stream factor of p53. All these results indicate that IL-7 regulate AMPK and mTOR via p53.

IL-7 inhibit autophagy of A549.
To confirm that IL-7 regulate autophagy suppression, western blot were used to analyze LC3-II/I protein level andTEM were applied to observe autophagosome as the group previously  www.nature.com/scientificreports/ designed: negative control group, cells treated with IL-7, IL-7 + A-IL-7R, Rapamycin, Rapamycin + IL-7 Compound C, Compound C + IL-7, PFT-α, and IL-7 + PFT-α. Western blot for LC3 show that IL-7 decrease LC3II/I protein level and this can be blocked by A-IL-7R (Fig. 5a). In addition, PFT-α and Rapamycin increase the LC3II/I (Fig. 5b,c) and Compound C decrease the LC3II/I level (Fig. 5d). Similar results were obtained in TEM  www.nature.com/scientificreports/ assay: IL-7 group, Compound C group and IL-7 + Compound C group find out a decrease of autophagosome; Rapamycin group, Rapamycin + IL-7 group, PFT-α and IL-7 + PFT-α group observed a noteworthy increase of autophagosome; control group observed an basic autophagosome level in A549 cells and the autophagosome level of IL-7 + A-IL-7R group is similar to control group (Fig. 6). Both the LC3 and TEM results indicate IL-7 inhibit autophagy of A549 cell line.  Fig. 7). Clinically, compared to the NSCLC with high expression of AMPK, tumors with low expressions of AMPK were more advanced-stage cancer (P < 0.001).
The expression levels of P53 and AMPK protein were not associated with a patient's age or sex, the tumor histological type, and differentiation (Tables 1, 2). In the Cox regression multivariate analysis, IL-7R, mTOR, and tumour stage were the strongest predictors of survival (Table 3).

Discussion
Autophagy plays dual role in cellular activities. On the one hand, by degrading and reusing damaged cellular organelles, autophagy maintain cellular survival and metabolism 6 . Some cancer cell lines, such as pancreatic cancer cells, have abnormally high autophagy level 6 . It is likely that autophagy is required for cancer aggression 14 . On the other hand, BECN1 (Beclin-1), an autophagy associated gene which play a role in autophagosome formation, are frequently deleted in some cancer, such as breast cancer, ovarian and prostate cancer 15 . Other autophagy proteins are observed aberration and expression alteration. There is an emerging hypothesis that autophagy may act as anticarcinogenesis in the beginning status of oncocytoma 15 , but once tumor is established, autophagy may increase for cancer cell survival and fight against stress 15,16 .
The role of IL-7 in tumor apoptosis, migration, proliferation and lymphangiogenesis has been reported. But its role in autophagy are still elusive. In a recent study, Jifeng Zhu etc. revealed that IL-7 inhibit macroautophagy in schistosome infected mice and this effect of IL-7 may exacerbated liver pathology 17 . In our latest study, we find IL-7 suppress formation of autophagosome in NSCLC, mainly by regulating AMPK/mTOR signaling pathway via translocation of nuclear/cytoplasmic p53 and regulating PI3K/AKT/mTOR signaling pathway via Beclin-1 18 . In addition, the effect of IL-7 could be blocked by A-IL-7R antibody.
Autophagy or glucose restriction leads to p53 degradation or suppression, but this effect are associated with p53 status 9,19 . Interestingly, there is also evidence suggest that p53 activate autophagy, for p53 directly or indirectly targeting autophagy related gene such as DRAM, ULK1 and Atg7 9 . Cytoplasmic p53 inhibit augophagy while nuclear p53 promote autophagy 6 . But the mechanism of how does different status of p53 affect autophagy are not known. To further interrogate the effect of different p53 status on the autophagy modulation, A549 cell line  www.nature.com/scientificreports/ (p53 wild type) were chose. Previously, we reported that IL-7 regulate autophagy by PI3K/AKT/mTOR signaling pathway via Beclin-1 18 and prevent apoptosis via p53 20 . IL-7 caused up-regulation of p-mTOR and change of p53. Thus, we asked whether p53 get involved in IL-7 regulated mTOR pathway and autophagy. By isolation of cytoplasmic and nuclear protein of p53 we find IL-7 cause p53 translocation from nucleus to cytoplasm at 12 h. we also observed an increase of p53 mRNA level by using RT-qPCR. Despite seldom do we observed suppression in p53 nuclear protein, the p53 mRNA level and p53 cytoplasmic protein do escalated. We speculate that p53 protein mainly express in nucleus in NSCLC, just a small part locate in cytoplasm. Thus a slightly shut down of p53 protein in nucleus were not detected by western blot explicitly, but a mildly increase in cytoplasm were observed. An increase of p53 mRNA validate IL-7 induce p53 expression. Then, by detecting total protein of AMPK and mTOR and its phosphorylated form, we observed IL-7 caused decrease of p-AMPK and increase of p-mTOR. Mechanistically, several molecular, such as AMPK, DRAM, DAPK-1, PTEN, IGF-BP3 have been reported as downstream factor of p53 21 . It is reported that some kinases, including CAMKK, LKB1, TGF-β-activated protein kinase 1 (TAK1), and ataxia telangiectasia mutated (ATM) kinase play a role as upstream factor of AMPK 22 . However, whether p53 regulate AMPK or AMPK affect p53 are still elusive 23 . mTOR is a core component of several distinct complexes including mTOR complexes 1 (mTORC1), mTOR complexes 2 (mTORC2), and a putative mTOR complexes 3 (mTORC3). These complexes interact with several kinase and protein such as AMPK, PI3K-Akt, serum and glucocorticoid-induced kinase (SGK) 24 . mTOR participated in many cellular behaviors, such as autophagy, apoptosis, transcription, as well as cell growth, cell proliferation, survival 25 . Our experiment conducted a serial of inhibitor to substantiate the relationship between p53, AMPK and mTOR. After incubated with PFT-α (p53 inhibitor), western blot results show that both nuclear and cytoplasmic p53 protein decreased, then we observed increase of p-AMPK expression level and decrease of p-mTOR level. Meanwhile, PFT-α block the effect of IL-7-regulated autophagy suppression. These results elucidate IL-7 regulate p53 translocate from nucleus to cytoplasm and p53 affect AMPK and mTOR. Compound C (AMPK inhibitor) could up-regulate In the 20,000×, the black arrow indicate characteristic autophagosome in A549 cells after treated with IL-7 (autophagosome decrease); cells treated with pifithrin-α (autophagosome notably increase); cells treated with pifithrin-α + IL-7 (autophagosome increase); cells treated with Compound C (autophagosome decrease); cells incubated with Compound C + IL-7 (autophagosome decrease); cells treated with A-IL-7R antibody + IL-7 (effect of IL-7-induced autophagosome depression was blunt); cells incubated with Rapamycin (autophagosome increase) and cells treated with IL-7 + Rapamycin (autophagyosome slightly increase).   www.nature.com/scientificreports/ p-mTOR expression and inhibit autophagy, but p53 protein level was not affected, which indicate AMPK is downstream factor of p53. Rapamycin (mTOR inhibitor) cause p-mTOR attenuation and autophagy activation, but had no effect on p53 and AMPK, illustrating p53 and AMPK regulate mTOR. These results demonstrated IL-7 inhibit autophagy via p53 regulated AMPK/mTOR signaling pathway in A549 cell line. LC3, the mammalian homologue of yeast Atg8, is localized in autophagosome membrane and commonly considered as autophagy marker 25,26 . But sometimes an increase in autophagosome does not correlate with LC3 aggregate 27 . Thus TEM is another reliable way to assess autophagy level. In our work, both LC3II/I protein level and TEM results indicate that IL-7 inhibit autophagy in A549 cell, and these effect can be blocked by A-IL-7R. In addition, PFT-α notably activate autophagy demonstrating that p53 suppressing lead to autophagy activation. In line with this, mTOR inhibition also cause autophagy induction. Besides, AMPK inhibiting cause autophagy suppression.
In our study, immunohistochemical analysis of 123 NSCLC specimens show that AMPK low expression are associated with p53, mTOR, IL-7, IL-7R high expression. Clinically, patients with AMPK low expression are more associated with advanced-stage cancer (P < 0.001) and patients with high expression of AMPK had a statistically significant longer survival than those with low expression of AMPK. Similar to our findings, Nan Li et al. find out in both squamous cell carcinoma and lung adenocarcinoma, p-AMPK low expression had a higher survival rate 28 . However, Daohui Gong et al. demonstrated that AMPKα1 was highly expressed in NSCLC cancer tissues and correlated with poor prognosis in patients with NSCLC 29 . The immunohistochemical analysis of p53 showed the opposite results with AMPK. Zhihua Teng et al. believe that lung cancer with p53-negative expression had a longer 3-year survival rate than those with p53-positive expression 30 . Cox regression multivariate analysis show IL-7R, mTOR, and tumor stage were independent predictors of survival.
In conclusion, we demonstrated that IL-7 induced p53 translocation from nucleus to cytoplasm then regulating AMPK/mTOR signaling pathway to inhibit autophagy. Expression of AMPK and P53 correlate with IL-7/ IL-7R level, clinical stages, and NSCLC patient survival. Together with other pro-tumorigenesis effect such as pro-proliferation, promote lymphangio -genesis, and anti-apotosis that IL-7 played in NSCLC. There still many problem to be solved. In our further study, we will investigate the interaction and balance between these cellular behavior and explore the feasibility for targeting IL-7 and combined with other inhibitor in NSCLC therapy.

Data availability
The data that support the findings of the present study are available from the corresponding author on reasonable request.